Project Summary Wnts serve as a conserved family of intercellular signaling ligands in the nervous system regulating synaptic development, neurotransmission strength and activity-dependent plasticity. Such multifaceted Wnt functions require tight management throughout neurodevelopment, as well as the ability to respond to acute changes in neuronal activity at maturity. The founding Wnt, Wingless (Wg), was discovered in the Drosophila genetic system, together with multiple Wnt regulators. Wg signaling is known to play critical roles in the development and activity- dependent plasticity of the Drosophila neuromuscular junction (NMJ) glutamatergic model synapse but most Wg regulator pathways have not been investigated at all in the context of synaptic mechanisms. The goal of this research project is to test the roles of two secreted, extracellular Wg regulators in synaptogenesis. The first targeted regulator is a secreted soluble heparan sulfate proteoglycan (HSPG) identified in the Drosophila wing disc, called Carrier of Wingless (Cow). In the developing wing, Cow binds Wg to coordinate extracellular distribution and intercellular signaling. However, Cow has not been studied in the nervous system, and nothing is known about its function at the synapse. The second regulator is a secreted extracellular deacylase also identified in the Drosophila wing disc, called Notum. In the developing wing, Notum restricts Wg signaling by cleaving an essential palmitoleate moiety from Wg, thus rending it unable to bind to its receptor. However, Notum has not been studied in the nervous system, and nothing is known about its function at the synapse. Utilizing powerful Drosophila genetic tools, this project will investigate the roles of Cow and Notum in both structural and functional NMJ synaptogenesis using characterized null mutants and cell-targeted RNAi knockdowns. Structure will be tested with confocal and transmission electron microscopy, and function will be tested with electrophysiology and live dye imaging. Preliminary results show that both Cow and Notum act at the synapse to strongly modulate synapse growth, structural architecture and functional differentiation. Both Cow and Notum will be tested as modulators of Wg trans-synaptic signaling by assaying synaptic Wg ligand levels and Wg- dependent signal transduction, as well as the downstream synaptic molecular composition. Preliminary results show upregulated Wg trans-synaptic signaling and consistently misregulated synaptic molecular assembly in both cow and notum mutants. The roles of elevated Wg signaling in both cow and notum mutant phenotypes will be determined by genetically correcting Wg trans-synaptic signaling, and then testing synapse structure, function and molecular composition. The overall goal of this work is to generate a deeper understanding of Wnt signaling regulation at neuromuscular and glutamateric synapses, by identifying control mechanisms acting in synaptogenesis and activity-dependent modulation. Identified pathways are expected to be critical in both normal and disease state synaptic mechanisms. A long-term goal of this work is to identify new therapeutic avenues for treatment of neurological disorders in which Wnt signaling goes awry.